Pretreatment method for extraction of nucleic acid from biological samples and kits therefor

ABSTRACT

The present invention relates to methods for pretreating biological samples for extraction of nucleic acid therefrom. The present invention employs a combination of at least one protein denaturant with one or more of the following elements to form a reaction mixture for extraction of nucleic acid: (1) at least one aprotic solvent, (2) stepwise heating, and (3) sample dilution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/359,854, filed Jan. 26, 2009, which is a continuation of U.S.application Ser. No. 10/359,179, filed Feb. 6, 2003, now, U.S. Pat. No.7,601,491, the disclosures of which are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods of treating biological samples,such as plasma and blood samples, for analysis. More specifically, theinvention relates to biological sample processing methods that arecompatible with subsequent nucleic acid analysis, such as hybridization,amplification and detection.

BACKGROUND OF THE INVENTION

Nucleic acid-based genetic methods for identification of microorganismshave greatly reduced the time and labor involved in clinical diagnosis.Such methods include, for example, nucleic acid hybridization (e.g.,Southerns/microarrays and slot blots), nucleotide sequencing, nucleicacid cloning techniques, restriction digestion of nucleic acids andnucleic acid amplification. In particular, nucleic acid amplificationhas provided means for rapid, sensitive and specific identification ofmicroorganisms by amplification and detection of specific genes or genefragments. For use as diagnostic methods, it is of particular interestto apply these nucleic acid analyses to biological samples such asplasma and whole blood samples. Prior to the availability of nucleicacid-based methods for detection and identification of microorganisms,plasma or blood samples were analyzed for the presence of microorganismsby blood culturing. However, processing of clinical samples for nucleicacid analyses requires different criteria than sample processing forculturing. For example, nucleic acids must be released from themicroorganism in a form suitable for the analysis; nucleic acids must bepresent in a composition with the appropriate components, ionic strengthand pH for the biochemical reactions of the analysis; and inhibitors ofthe reactions such as nucleases, if present in the clinical sample orintroduced during sample processing, must be removed or renderednon-inhibitory.

One potential biochemical detection method involves the use of nucleicacid hybridization. The sequence specificity embodied in nucleic acidsmakes it possible to differentiate virtually any two species by nucleicacid hybridization. Standard techniques for detection of specificnucleotide sequences generally employ nucleic acids that have beenpurified away from cellular proteins and other cellular contaminants.The most common method of purification involves lysing the cells withsodium dodecyl sulfate (SDS), digesting with proteinase K (ProK), andremoving residual proteins and other molecules by extracting withorganic solvents such as phenol, chloroform, and isoamylalcohol.

Endogenous nucleases released during cell solubilization can frustrateefforts to recover intact nucleic acids, particularly ribonucleic acids(RNA). While deoxyribonucleases (DNases) are easily inactivated by theaddition of chelating agents to the lysis solution, ribonucleases(RNases) are far more difficult to eliminate. RNases are ubiquitous,being present even in the oil found on human hands. Accordingly,protecting against RNase is a commonly acknowledged aspect of anystandard RNA preparation technique. The standard procedure for preparinglaboratory stocks of pancreatic RNase is to boil a solution of theenzyme for 15 minutes. The purpose of this treatment is to destroy alltraces of contaminating enzyme activity because other enzymes cannotsurvive boiling.

Sambrook, et al., Molecular Cloning, 3^(rd) Edition (2001), a compendiumof commonly followed laboratory practices, recommends extensiveprecautions to avoid RNase contamination in laboratories. Suchprecautions include preparing all solutions that will contact RNA usingRNase-free glassware, autoclaved water, and chemicals reserved for workwith RNA that are dispensed exclusively with baked spatulas. Besidespurging laboratory reagents of RNase, RNase inhibitors are typicallyincluded in lysis solutions. These are intended to destroy endogenousRNases that generally become activated during cell lysis. Also, it iscommon practice to solubilize RNA in diethyl pyrocarbonate(DEPC)-treated water. Moreover, in an attempt to improve the handling ofRNA samples, formamide has been tested as a solubilizing agent for thelong-term storage of RNA. Chomczynski, P., Nucleic Acids Research 20,3791-3792 (1992).

Protecting against RNase is cumbersome and costly, and typicalextraction procedures require the handling of caustic solvents, accessto water baths, fume hoods, and centrifuges, and even the storage anddisposal of hazardous wastes. The direct analysis of unfractionatedsolubilized biological samples would avoid the cost and inconvenience ofthese purification techniques.

In view of the foregoing, there exists a need for a simple and rapidmethod by which biological samples such as plasma and blood may betreated for the extraction therefrom of nucleic acid for analysis.

SUMMARY OF THE INVENTION

The present invention addresses the need for a simple and rapid methodto treat biological samples such as plasma and blood for extraction ofnucleic acid therefrom. In one embodiment, the method of the presentinvention pretreats biological samples for extraction of nucleic acidtherefrom by mixing the biological samples with at least one proteindenaturant and by stepwise heating the mixture in a temperature range offrom about 55° C. to about 85° C. to form a reaction mixture. In anotherembodiment, the method of the present invention pretreats the biologicalsamples by mixing the biological samples with at least one proteindenaturant in a temperature range from about 55° C. to about 85° C. toform a reaction mixture and by diluting the reaction mixture with anaqueous solution or an aprotic solvent. Yet in another embodiment, themethod of the present invention pretreats biological samples forextraction of nucleic acid therefrom by treating the samples with atleast one protein denaturant and at least one aprotic solvent at orabove about 4° C. to form a reaction mixture for nucleic acid analysis.In a preferred embodiment, the method of the present invention pretreatsbiological samples by treating the samples with at least one proteindenaturant and at least one aprotic solvent at or above about 25° C. Inan exemplary embodiment of the present invention, ProK is used as theprotein denaturant, formamide is used as the aprotic solvent, and thetreatment is conducted at a temperature in the range of about 55° C. toabout 85° C. for about 30 minutes.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “purifying” and “purification” also includeextracting/extraction and isolating/isolation.

The present invention is a composition and method of treating biologicalsamples such as, for example, plasma and whole blood samples, forextraction of nucleic acid therefrom. The present invention employs acombination of at least one protein denaturant with one or more of thefollowing elements to form a reaction mixture for extraction of nucleicacid: (1) at least one aprotic solvent, (2) stepwise heating, and (3)sample dilution.

The biological samples used according to the present invention may beany biological material containing nucleic acid such as, for example,clinical, forensic or environmental samples. These samples may containany viral or cellular material, including prokaryotic and eukaryoticcells, viruses, bacteriophages, mycoplasms, protoplasts and organelles.Such biological materials may comprise all types of mammalian andnon-mammalian animal cells, plant cells, algae including blue-greenalgae, fungi, bacteria, yeast and protozoa. Representative examplesinclude blood and blood-derived products such as whole blood, plasma andserum; clinical specimens such as semen, urine, feces, sputa, tissues,cell cultures and cell suspensions, nasopharangeal aspirates and swabs,including endocervical, vaginal, occular, throat and buccal swabs; andother biological samples such as finger nails, skin, hair andcerebrospinal fluid or other body fluid.

The protein denaturant is a reagent that is capable of, by itself orwhen combined with other protein denaturants and/or aprotic solvents,disrupting the protein membranes or walls of cells, virions, DNase orRNase, causing protein denaturation and organism lysis and releasingnucleic acid in biological samples. Protein denaturants are well knownin the art, and may be purchased from known vendors or prepared usingwell-known standard techniques. Protein denaturants that are useful inthe present invention include proteolytic enzymes, such as ProK,pronase, pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase;anionic, non-ionic and zwitterionic detergents such as SDS, lithiumdodecyl sulfate (LDS), polyethylene glycol sorbitan monolaurate (i.e.,Tween® 20), polyethylene glycol sorbitan monooleate (i.e., Tween® 80),NP-40, dodecyl trimethyl ammonium bromide (DTAB), cetyl trimethylammonium bromide (CTAB),3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),polyethylene glycol tert-octylphenyl ether (i.e., Triton X detergentssuch as Triton X-20 and Triton X-100); surfactants such as surfactin;solvents such as phenol, chloroform, and isoamylalcohol; amides such asN-ethylacetamide, N-butylacetamide and N,N-dimethyl-acetamide; reducingagents such as glutathione, β-mercaptoethanol and dithiothereitol (DTT);protein denaturing salts such as NaCl, KCl, LiCl, NH₄Cl, (NH₄)₂SO₄ andperchlorate salt; and agents that cause an increase in pH such as KOH,NaOH, NH₄OH and Ca(OH)₂. Proteolytic enzymes are generally preferred asthe protein denaturant, and ProK is exemplary of such proteolyticenzymes. The usefulness of any protein denaturant in the method of thepresent invention may be readily ascertained by one skilled in the artusing routine screening methods that do not require undueexperimentation.

When used in the methods of the present invention, the concentration ofthe protein denaturants can vary depending on other agents andconditions, but is sufficient to disrupt the protein membranes or wallsof cells, virions, DNase or RNase, causing protein denaturation andorganism lysis and releasing nucleic acid in biological samples in thepresence of other protein denaturants and/or aprotic solvents. Thisconcentration of protein denaturant can be readily determined by thoseskilled in the art using routine screening methods that do not requireundue experimentation. When ProK is used as the protein denaturant withat least one aprotic solvent, the desirable concentration of ProKdepends on the proteinaceous content of the biological samples. For mostbiological samples, it is in the range of about 1 to about 100 units permilliliter of biological sample. However, some biological samples mayrequire dilution or concentration prior to the pretreatment in order tomake use of ProK in its optimal concentration range, i.e., about 1 toabout 100 units per milliliter of biological sample. When a base or asalt is used as the protein denaturant, for most biological samples, theconcentration of the base or salt is in the range of about 10 to about400 mM, more preferably about 80 to about 220 mM, and most preferablyabout 100 mM. When a detergent is used as the protein denaturant, formost biological samples, the concentration of the detergent is in therange of about 0.05% to about 8.0%, more preferably about 0.05% to about4%, and most preferably about 1%.

More than one protein denaturant can be utilized for the pretreatmentaccording to the present invention. The combinations of the same type ordifferent types of protein denaturants offer additional advantages inthe pretreatment of biological samples for the extraction of nucleicacids. The concentrations of mixed protein denaturants in the method ofthe present invention can also be readily ascertained by one skilled inthe art using routine screening methods that do not require undueexperimentation.

In one embodiment, one or more aprotic solvents are utilized with theprotein denaturant(s). The aprotic solvent used in the present inventionis capable of dissolving ionic substances because of the permanent orinduced dipole, which allows the formation of an ion-dipole force.Aprotic solvents do not donate suitable hydrogen atoms to form labilehydrogen bonds with anions. Nucleophilic substitution by the SN2mechanism with a charged nucleophile is often faster in aproticsolvents.

The dipolar aprotic solvent is a solvent with a comparatively highrelative permittivity (or dielectric constant), e.g., greater than about15, and a dipole moment. However, unlike water, the more common polarsolvent, aprotic solvents do not ionize to form hydrogen ions, whichconfers advantages.

Aprotic solvents useful in the present invention include formamide,dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide(DMAC), acetronitrile, benzene, toluene, acetone, cyclohexane,n-heptane, sulfur dioxide, hexamethylphosphoramide (HMPA) and othernon-aqueous media that can be used to denature and solubilize the targetnucleic acid. Formamide is a preferred aprotic solvent. Such aproticsolvents are well known in the art and may be purchased from knownvendors or synthesized using well-known standard techniques. Theusefulness of any aprotic solvent in the method of the present inventionmay be readily ascertained by one skilled in the art using routinescreening methods that do not require undue experimentation.

When used in the method of the present invention, the concentration ofthe aprotic solvent can vary depending on other agents and conditions,but is sufficient to protect a nucleic acid by maintaining the nucleicacid in solution at the required temperature and in the presence of atleast one protein denaturant. This concentration can be readilydetermined by those skilled in the art using routine screening methodsthat do not require undue experimentation. When formamide is the aproticsolvent used in the method of the present invention, for most biologicalsamples, the concentration of formamide is preferably in the range ofabout 10% to about 80% by volume of the reaction mixture, morepreferably about 20% to about 40%, and most preferably about 30%.

More than one aprotic solvent can be utilized for the pretreatmentaccording to the present invention. The combinations of differentaprotic solvents offer additional advantages in the pretreatment ofbiological samples for the extraction of nucleic acid therefrom. Theconcentrations of mixed aprotic solvents in the methods of the presentinvention can also be readily ascertained by one skilled in the artusing routine screening methods that do not require undueexperimentation.

The temperatures at which the methods of the present invention areconducted are generally described as at or above about 4° C., preferablyat or above about 25° C. A more preferred range is from about 55° C. toabout 95° C. An even more preferred range of temperature is about 65° C.to about 85° C. The most preferred temperature is about 70° C. It isbelieved that the high temperature contributes to the denaturation ofproteins in the method of the present invention.

In another embodiment of the invention, stepwise heating is utilizedwith protein denaturant(s) alone or in combination with aproticsolvent(s) for extraction of nucleic acid from biological samples.Stepwise heating is a heating procedure that increases or decreases thetreatment temperature systematically by two or more steps for theextraction of nucleic acid from biological samples. For example,twenty-minute treatments at each treatment temperature of 55° C. and 85°C. are utilized for the protein denaturation and nucleic acid extractionwith the proteolytic enzyme, Pro K. Stepwise heating has shown improvedrecoveries for nucleic acid extraction. The temperature ranges and theduration of the heating steps in the method of the present invention canbe readily ascertained by one skilled in the art using routine screeningmethods that do not require undue experimentation.

In yet another embodiment of the present invention, sample dilution withan aqueous solution or an aprotic solvent is utilized with proteindenaturant(s) alone or with aprotic solvent(s). The aqueous solution canbe water or any buffer solution with a pH value between about 3.0 toabout 10.0. The sample dilution has shown improved recoveries fornucleic acid extraction. The dilution step brings about further proteindenaturation and/or precipitation while maintaining the nucleic acid insolution. The dilution factor (reaction mixture/diluent) is usuallybetween 4:1 and 1:10 depending on the sample, the diluent and thetreatment condition. The choice of diluent and dilution factor for usein the method of the present invention can be readily determined by oneskilled in the art using routine screening methods that do not requireundue experimentation.

Yet another aspect of the present invention is to provide kits fortreating a biological sample for the extraction of nucleic acidtherefrom, wherein the kits comprise at least one protein denaturantwith or without one or more aprotic solvents as described herein. Thekits may contain water and buffer solutions as described herein, as wellas iron oxide or other solid supports for nucleic acid purification,which are described in more detail elsewhere. The kits may also containone or more of the following items for processing and assaying thebiological samples: collection devices such as swabs, tubes andpipettes; controls; pH indicators; and thermometers. Kits may includecontainers of reagents mixed together in suitable proportions forperforming the method in accordance with the present invention. Reagentcontainers preferably contain reagents in unit quantities that obviatemeasuring steps when performing the subject method.

The present invention also includes the reaction mixtures, as well asmethods of extracting nucleic acid from the reaction mixtures. Thereaction mixtures comprise at least one protein denaturant and with orwithout one or more aprotic solvents. The reaction mixtures may in someembodiments include various reagents used with the subject reactionmixtures to purify and detect nucleic acids, such as buffers and ironoxide or other solid supports for nucleic acid purification.

The invention will now be described in greater detail by way of thespecific examples. The following examples are offered for illustrativepurposes and are not intended to limit the invention in any manner. Inthese examples, the reversible binding of nucleic acid molecules onparamagnetic particles in an acidic environment, as disclosed in U.S.Pat. No. 5,973,138 to Collis, which is incorporated herein by reference,is used for nucleic acid isolation from the reaction mixture resultingfrom treating samples for extraction of intact nucleic acid according tothe present invention. The binding pH is preferably about 1 to about6.5, more preferably about 1 to about 4, and most preferably about 2.The elution pH is preferably about 6.5 to about 12, more preferablyabout 7.5 to about 11, and most preferably about 8. The paramagneticiron oxide technology captures nucleic acids non-specifically, orindependent of sequence. There are several other automated nucleic acidextraction technologies currently on the market, representing bothspecific and non-specific capture. The most notable non-specific capturesystems include the Roche MagNA Pure LC and the Organon TeknikaNuclisens, both of which utilize magnetic silica particles. The QiagenBioRobot 9604 incorporates silica membranes. The Roche AmpliPrepcaptures targets specifically with streptavidin-coated magneticparticles and biotinylated capture probes. GenProbe's Tigris system isexpected to utilize oligonucleotide-coated magnetic particles. Inaddition, materials such as iron oxide, silica-coated particles,silica-coated membranes, glass fiber mats, glass membranes and otherglasses, zeolites and ceramics can also be used as a solid phase bindingsurface for nucleic acid extraction. In summary, any conventionaleffective technique for nucleic acid isolation and purification known inthe art, including liquid and solid phase separation, can be utilizedfor the isolation and purification of nucleic acids following thepretreatment process of the present invention or, alternatively, thepretreatment process may be performed in the presence or absence ofconventional effective techniques for nucleic acid isolation andpurification that are known in the art.

Strand displacement amplification (SDA) is also utilized in theseexamples for amplification and detection of the target nucleic acidsequence following the extraction process of the invention and anyappropriate isolation or purification step. The SDA method involvesfirst mixing single-stranded target sequences with a nucleic acidpolymerase, restriction endonuclease, deoxynucleoside triphosphates andat least one primer, which is complementary to a region at the 3′ end ofa target fragment, wherein each primer has a sequence at the 5′ end thatis a recognition sequence for a restriction endonuclease, and thenallowing the mixture to react for a time sufficient to generate reactionproducts. Where the nucleic acids comprise RNA, it is preferable to usereverse transcriptase to convert RNA to cDNA. The invention, however, isnot limited to SDA detection, and many conventional and effectivedetection techniques such as hybridization and polymerase chain reaction(PCR) may be used for detection following the pretreatment process ofthe invention.

The following examples illustrate the effectiveness of the pretreatmentprocess of the present invention to pretreat whole blood and plasmasamples for nucleic acid extraction. Whole blood and plasma are amongthe most challenging samples for nucleic acid extraction because oftheir highly proteinaceous content; therefore, the methods of thepresent invention are expected to be effective for other biologicalsamples as well. Representative examples are discussed herein.

Example 1 Evaluation of Plasma Pretreatment at Varying Temperatures

The experiment was designed to evaluate the effect of temperature duringthe ProK plasma pretreatment of the present invention on extraction ofRNA. The RNA was extracted from the sample using iron oxide.

First, 40 mg of iron oxide and 1200 uL of 30 mM potassium phosphatebuffer (KPB) were dispensed into eight 2 mL polypropylene tubes. Plasma(600 uL), anti-coagulated with EDTA was added to six of the tubes, and600 uL of 30 mM KPB was added to the remaining two tubes. Three units ofProK were then added to each tube, and the tubes were mixed. The twotubes containing KPB and two of the tubes containing plasma wereincubated at room temperature for 20 minutes. Two tubes containingplasma were incubated at 37° C. for 20 minutes, and two tubes containingplasma were incubated at 52° C. for 20 minutes. Following incubation,180 uL of 6 M glycine/HCl, 1 ug of carrier RNA and 5000 HIV in vitrotranscripts were added to each tube. The tubes were then mixed for 15minutes by alternately turning electromagnets, positioned on oppositesides of the tubes, on and off. This mixed the samples by drawing theiron oxide particles back and forth through the solution. The iron oxideparticles were then magnetically locked to the sides of the tubes byturning on the electromagnets. The unbound sample from each tube wasthen removed by aspiration. The particles were then washed twice with 2mL of 90 mM glycine/HCl. The tubes were then mixed, the particles werelocked to the side, and the fluid removed by aspiration as describedabove. The samples were eluted from the iron oxide in each tube byadding 0.4 mL of elution buffer composed of 45 mM KOH, 90 mM Bicine, and20 mM KPO₄ and mixing the tubes. Following elution, the eluents weretransferred to new tubes by magnetically locking the iron oxideparticles to the side and aspirating the sample into new tubes. Next, 1ug of yeast carrier RNA was added to each tube. The eluted samples werethen assayed by SDA using an HIV reverse transcriptase (RT)-SDA assaysystem. The results were as follows:

Plasma (uL) Temperature Mean Signal 0 25° C. 95,969 600 25° C. 41,410600 37° C. 5,896 600 52° C. 46,709

The signal responses at various temperatures indicate RNA targetrecoveries when plasma is pretreated with ProK at temperatures as highas 52° C.

Example 2 Evaluation of Plasma Pretreatment at High ProK Concentrationand Temperature

The following experiment examined the effect of varying ProKconcentrations and high temperature plasma pretreatment on extraction ofRNA. The samples were extracted using iron oxide.

EDTA anti-coagulated plasma (600 uL) collected in PPT™ tubes from BectonDickinson (BD) was transferred to new tubes, each containing 40-45 mgiron oxide. Following transfer, 220 uL of 30 mM KPB was added to each ofthe tubes. Next, 3, 6, or 9 units of ProK was added to the tubes, andthe tubes were then incubated for 20 minutes in a 55° C., 65° C., or 75°C. water bath. Following incubation, 180 uL of 6 M glycine/HCl was addedto each tube, and the tubes were mixed by aspirating up and down with apipette. Yeast carrier RNA (10 uL of 10 ug/mL) was added to each tube.The samples were then spiked with 6 uL of 10⁷ copies/mL HIV RNA in vitrotranscript. The samples were mixed by aspirating and dispensing 800 uLat a time, repeated 24 times. Following mixing, the samples were thenextracted as described in Example 1, i.e., by magnetically locking theiron oxide particles to the sides of the tubes and aspirating theunbound samples. The particles were washed twice with 1 mL of 86 mMglycine/HCl by aspirating and dispensing 800 uL for a total of 12 times,locking the iron oxide particles to the side of the tubes, andaspirating the fluid from tubes. The sample was then eluted from theiron oxide by adding 0.4 mL of elution buffer composed of 90 mM Bicine,50 mM KOH, and 20 mM KPO₄ to each tube, mixing by aspirating anddispensing 300 uL 12 times. Following elution, 10 microliters of yeastcarrier RNA (10 mg/mL) was added to each tube. The tubes were thenheated to 60° C. while being subjected to magnetic mixing for 20 minutesby alternately turning electromagnets, positioned on opposite sides ofthe tubes, on and off. This mixed the samples by drawing the iron oxideparticles back and forth through the solution. The eluted samples weretransferred to new tubes as described in Example 1, i.e., bymagnetically locking the iron oxide particle to the side and aspiratingthe sample into new tubes and, assayed by SDA using an HIV reversetranscriptase (RT)-SDA assay system. The results are as follows:

Sample Temperature Proteinase K Mean Signal Clean 55° C. 9 U 2,883 65°C. 6 U 54,824 75° C. 3 U 71,577 Plasma 55° C. 3 U 1,759 6 U 13,611 9 U12,356 65° C. 3 U 8,241 6 U 23,029 9 U 20,961 75° C. 3 U 17,650 6 U12,767 9 U 19,554

The results demonstrate that the pretreatment of plasma with 6-9 unitsof ProK results in target recovery throughout the temperature rangetested (55° C. to 75° C.). Furthermore, the correlation between thetemperature and the amount of ProK shows that at higher temperatures,target recovery can be achieved throughout the ProK range tested (3-9units of ProK).

Example 3 Evaluation of Plasma Pretreatment with Formamide and VaryingThermal Profiles

The following experiment examined the effect of ProK and formamide andvarying thermal profiles during plasma pretreatment on extraction ofRNA. The RNA was extracted from the sample using iron oxide.

First, 500 uL of EDTA anti-coagulated plasma was added to 64 tubes, eachcontaining 40-45 mg of iron oxide. Eight of these tubes were set asideas controls, i.e. were not pretreated. Phosphate buffered saline (PBS)(300 uL) was added to eight of the tubes. The remaining forty-eighttubes received 300 uL of formamide. To each of the fifty-six tubes thatcontained either PBS or formamide, 20 units of ProK was added. The tubeswere incubated at the temperatures and times specified in the summarytable. All of the tubes were then spiked with 2,500 copies of HIV invitro transcript to simulate a plasma sample containing 5,000 copies/mLHIV RNA. Next, 180 uL of 6 M glycine/HCl was added to each tube, and thesamples were mixed by aspirating 800 uL up and down with a pipette for atotal of 24 times. The iron oxide particles were then magneticallylocked to the sides of the tubes, and the unbound samples were removedby aspiration. The particles were washed twice with 1 mL of 90 mMglycine/HCl by aspirating and dispensing 800 uL for a total of 12 times.After each wash, the particles were locked to the sides of the tubes andthe fluid was removed by aspiration from each tube. The samples wereeluted from the particles with the addition of 120 uL of elution buffercomposed of 75 mM Bicine, 85 mM KOH and mixing by aspirating anddispensing 100 uL at a time for a total of 15 times. The samples werethen neutralized by adding 60 uL of neutralization buffer composed of400 mM Bicine to each tube and mixed by aspirating and dispensing 100 uLat a time for a total of 15 times. The eluted samples were transferredto new tubes as described in Example 1. The eluted samples were assayedby SDA using an HIV RT-SDA assay system. The results are as follows:

Mean Pretreatment Reagents Pretreatment Incubation(s) Signal None None8597 ProK/PBS 20 min. @ 75° C. 4398 ProK + Formamide 20 min. @ 85° C.37948 ProK + Formamide 20 min. @ 65° C. + 10 min. @ 70° C. 100894 ProK +Formamide 20 min. @ 65° C. + 10 min. @ 85° C. 63822 ProK + Formamide 30min. @ 70° C. 99491 ProK + Formamide 20 min. @ 70° C. 74560

The results clearly demonstrate that pre-treating plasma with ProK andformamide for the times and temperatures described gives significantlyhigher mean signals than not pretreating or by pretreating with ProK andPBS for 20 minutes at 75° C.

Example 4 Evaluation of DNA Extraction from Plasma, with and withoutIron Oxide Present During Plasma Pretreatment

The following experiment was conducted to compare DNA extractionefficiency from plasma when iron oxide is present during plasmapretreatment versus iron oxide being added after pretreatment.

Human plasma (500 uL) was added to each of twelve 2 mL tubes—sixcontaining 40 mg iron oxide and six empty tubes. ProK (5 units) wasadded to each tube, and the tubes were incubated for 20 minutes at 65°C. Formamide (400 uL) was added, and the tubes were incubated for 10minutes at 70° C. The samples that were pretreated without iron oxidewere transferred to six new 2 mL tubes containing 40 mg iron oxide.Next, 2,500 copies of K10 DNA plasmid containing an M. tuberculosis (TB)specific sequence were spiked into five of the six tubes that werepretreated without iron oxide present and into five of the six tubesthat were pretreated with iron oxide present. To each tube was added 180uL of 6 M glycine/HCl, followed by mixing by aspirating up and down. Theiron oxide particles were magnetically locked to the sides of the tubes,and the unbound samples were aspirated. Tubes were washed twice with 5mM glycine/HCl, locking particles to sides of tubes after each wash andaspirating fluid from tubes. Elution buffer (120 uL) composed of 105 mMKOH and 14% DMSO was added and mixed by pipetting up and down. Theeluted samples were then transferred to new tubes as described inExample 1. Neutralization buffer (60 uL) composed of 350 mM Bicine and38.5% glycerol was added and mixed by pipetting up and down. Elutedsamples were amplified in a Direct TB SDA assay (DTB) to obtain DTBspecific response and internal amplification control (IC) response. Theresults are as follows:

Pretreatment Mean DTB Mean IC Condition Target Signal Signal No ironoxide at   0 K10/sample 7 30639 pretreatment 2500 K10/sample 33810 2021440 mg iron oxide at   0 K10/sample 0 19898 pretreatment 2500 K10/sample2966 32178

The results demonstrate that combining plasma and iron oxide afterplasma pretreatment, rather than before pretreatment, improves DNAextraction efficiency from plasma as indicated by signal improvement inthe TB amplification assay.

Example 5 Evaluation of DNA Extraction from Plasma, with and withoutIron Oxide Present During Plasma Pretreatment, and with and without ACField Applied During Mixing

The following experiment was conducted to compare DNA extractionefficiency from plasma when iron oxide is present during plasmapretreatment versus when iron oxide was added after pretreatment and toexamine the two conditions with and without an alternating current (AC)field during mixing.

Human plasma (500 uL) was added to each of twenty-four 2 mL tubescontaining 40 mg iron oxide and to twenty-four empty 2 mL tubes. ProK (5units) and 300 uL formamide were added to each tube. All tubes wereincubated for 20 minutes at 65° C. and then for 10 minutes at 85° C. Thesamples that were pretreated without iron oxide were transferred to new2 mL tubes containing 40 mg iron oxide. Next, 4,000 copies of K10 DNAplasmid containing an M. tuberculosis (TB) specific sequence was spikedinto 20 of the 24 tubes that were pretreated without iron oxide presentand into 20 of the 24 tubes that were pretreated with iron oxidepresent. Then, 150 uL of 6 M glycine/HCl was added and mixed byaspirating up and down. Twelve of the tubes pretreated with iron oxidepresent and 12 of the tubes pretreated without iron oxide present weremixed without an AC field at each mix step. The remaining 12 tubespretreated with iron oxide present, as well as the remaining 12 tubespretreated without iron oxide present, were mixed under a 30 mV AC fieldat each mix step. The iron oxide particles were magnetically locked tothe sides of the tubes, and the unbound sample was aspirated from thetubes. The tubes were washed twice with 5 mM glycine/HCl, lockingparticles to sides of tubes after each wash and aspirating fluid fromtubes. Next, 120 uL of elution buffer composed of 105 mM KOH and 14%DMSO was added and mixed by pipetting up and down. The eluted sampleswere transferred to new tubes as in Example 1. Neutralization buffer (60uL) composed of 350 mM Bicine and 38.5% glycerol was added and mixed bypipetting up and down. Eluted samples were amplified in the Direct TBSDA assay to obtain DTB specific response and IC response. The resultsare as follows:

Pretreatment AC at Mean DTB Mean IC Cond. Mixing Target Signal Signal Noiron oxide None   0 K10/sample 0 45599 4000 K10/sample 41428 40049 30 mV  0 K10/sample 4 37409 4000 K10/sample 69126 33033 40 mg iron oxide None  0 K10/sample 0 51567 4000 K10/sample 10470 44495 30 mV   0 K10/sample0 32879 4000 K10/sample 7820 36261

The results demonstrate that combining plasma and iron oxide afterplasma pretreatment, rather than before pretreatment, improves DNAextraction efficiency from plasma as indicated by signal improvement inthe TB amplification assay. Mixing in the presence of the AC fieldimproved signal for samples that did not contain iron oxide duringpretreatment.

Example 6 Evaluation of the Effect of Diluting Plasma Sample Prior toProK Digestion Versus after ProK Digestion on DNA Extraction from Plasmawith Iron Oxide

The following experiment was conducted to compare DNA extractionefficiency from plasma with iron oxide when plasma is diluted prior toProK digestion with potassium phosphate buffer versus diluted after ProKdigestion with water.

Human plasma (500 uL) was added to each of twenty-four 2 mL tubes. ProK(5 units) and 400 uL of 30 mM potassium phosphate buffer were added toeight of the plasma tubes and incubated for 20 minutes at 65° C.followed by incubation for 10 minutes at 70° C. ProK (9 units) and 400uL of 30 mM potassium phosphate buffer were added to eight of the plasmatubes and incubated for 20 minutes at 65° C. followed by incubation for10 minutes at 70° C. ProK (5 units) was added to eight of the plasmatubes and incubated for 20 minutes at 65° C. Following the 65° C.incubation, these tubes were diluted with 400 uL of water and incubatedfor 10 minutes at 70° C. Potassium phosphate buffer (900 uL) was addedto the remaining eight 2 mL tubes. Each of the solutions weretransferred to new 2 mL tubes containing 40 mg iron oxide each. Next,3,000 copies of K10 DNA plasmid containing an M. tuberculosis specificsequence was spiked into six of the eight tubes of each of the fourconditions. The remaining two tubes of each condition were left asnegative controls. Next, 180 uL of 6 M glycine/HCl was added to eachtube and mixed by aspirating up and down. The iron oxide particles weremagnetically locked to the sides of the tubes, and the unbound sampleswere aspirated. The tubes were washed twice with 5 mM glycine/HCl,locking particles to the sides of tubes after each wash and aspiratingfluid from the tubes. Elution buffer (120 uL) composed of 105 mM KOH and14% DMSO was added and mixed by pipetting up and down. The elutedsamples were transferred to new tubes as described in Example 1.Neutralization buffer (60 uL) composed of 350 mM Bicine and 38.5%glycerol was added and mixed by pipetting up and down. Eluted sampleswere amplified in the Direct TB SDA assay to obtain DTB specificresponse and IC response. The results are as follows:

Mean DTB Mean IC Sample Dilution Target Signal Signal Buffer N/A   0K10/sample 0 43048 3000 K10/sample 28611 42912 Plasma Pre-ProK (5 U)   0K10/sample 15 48233 3000 K10/sample 2984 50713 Pre-ProK (9 U)   0K10/sample 1 46930 3000 K10/sample 13126 50987 Post-ProK (5 U)   0K10/sample 45 52543 3000 K10/sample 22893 49802

The results demonstrate that diluting the plasma following ProKdigestion improves DNA extraction efficiency from plasma as indicated byincreased signal in the TB amplification assay. Results from thepost-ProK dilution condition approach those of the buffer samplecontrol.

Example 7 Evaluation of Formamide and Water as Plasma Diluents in DNAExtraction with Iron Oxide

The following experiment was conducted to compare DNA extractionefficiency from plasma with iron oxide when plasma is diluted after ProKdigestion with either formamide or water.

To each of twelve 2 mL tubes was added 500 uL human plasma and 5 unitsof ProK. The tubes were incubated for 20 minutes at 65° C. Formamide(400 uL) was added to six of the tubes, and the tubes were incubated for10 minutes at 70° C. Water (400 uL) was added to the remaining sixtubes, and the tubes were incubated for 10 minutes at 70° C. Each of thesolutions were transferred to new 2 mL tubes containing 40 mg iron oxideeach. Next, 2,500 copies of K10 DNA plasmid, containing an M.tuberculosis specific sequence were spiked into five of the six tubes ofeach of the two conditions. The remaining tube of each condition wasleft as a negative control. Next, 180 uL of 6 M glycine/HCl was added toeach of the tubes and mixed by aspirating up and down. The iron oxideparticles were magnetically locked to the sides of the tubes, and theunbound samples were aspirated from the tubes. The tubes were washedtwice with 5 mM glycine/HCl, locking particles to the sides of tubesafter each wash and aspirating fluid from tubes. Elution buffer (120 uL)composed of 105 mM KOH and 14% DMSO was added and mixed by pipetting upand down. The eluted samples were transferred to new tubes as describedin Example 1. Neutralization buffer (60 uL) composed of 350 mM Bicineand 38.5% glycerol was added and mixed by pipetting up and down. Elutedsamples were amplified in the Direct TB SDA assay to obtain DTB specificresponse and IC responses. The results are as follows:

Mean DTB Mean IC Diluent Target Signal Signal Formamide   0 K10/sample 047601 3000 K10/sample 36305 41066 Water   0 K10/sample 0 50143 3000K10/sample 41650 28297

The results demonstrate that diluting the plasma following ProKdigestion with either water or formamide yields similar DNA extractionefficiency from plasma as indicated by similar signal in the TBamplification assay.

Example 8 Evaluation of Varying Carrier RNA Concentrations on Recoveryof RNA from Plasma with Iron Oxide

The following experiment was conducted to evaluate the effect ofincreasing yeast carrier RNA concentrations on RNA extraction fromplasma with iron oxide.

Human plasma was spiked with 10,000 HIV particles per mL, and 500 uL ofHIV-spiked plasma was added to forty 2 mL tubes. ProK (40 units) wasadded to each tube. Next, 300 uL of 30 mM KP containing 0, 2.5, 5, 10,or 20 ug carrier RNA was added to the tubes (eight tubes per carrier RNAlevel). Given a 0.5 mL plasma sample, this translates into carrier RNAconcentrations of 0, 5, 10, 20, and 40 ug per mL plasma. To serve as RNAcontrols, 500 uL non-spiked plasma, 40 units of ProK, 300 uL offormamide and 10,000 copies of HIV in vitro transcript were added toeight 2 mL tubes. All tubes were incubated for 30 minutes in a 70° C.water bath. Next, 180 uL of 6 M glycine/HCl was added and mixed byaspirating and dispensing 800 uL 24 times. The iron oxide particles weremagnetically locked to the sides of the tubes, and the unbound sampleswere aspirated. The tubes were washed twice with 1 mL of 6 mMglycine/HCl by aspirating and dispensing 800 uL 15 times. The particleswere locked to the side, and fluid was aspirated from the tubes. Elutionbuffer (120 uL) composed of 75 mM Bicine, 85 mM KOH, and 30 mM KPO₄ wasadded to each tube and mixed by aspirating and dispensing 100 uL 15times. The eluted samples were transferred to new tubes as described inExample 1. Neutralization buffer (60 uL) composed of 400 mM Bicine wasadded to each tube and mixed by aspirating and dispensing 100 uL 15times. The iron oxide particles were magnetically locked to the sides ofthe tubes to allow for removal of eluted sample. The eluted samples wereamplified in a HIV RT-SDA assay. The results are as follows:

Target CRNA/mL Plasma Mean Signal HIV Particles   0 ug 10577 HIVParticles 2.5 ug 12412 HIV Particles 5.0 ug 18008 HIV Particles 10.0 ug 18959 HIV Particles 20.0 ug  21910 HIV in vitro transcript   0 ug 21962

The results demonstrate that the presence of carrier RNA improvesrecovery of specific RNA target from plasma. Carrier RNA was previouslytitrated into the HIV amplification assay up to 20 ug and shown to haveno effect on amplification.

Example 9 ProK Pretreatment of Plasma with Stepwise Heating for NucleicAcid Extraction

The following experiment was conducted to examine the effects ofactivating ProK at 55° C. or 65° C. followed by a 20 minute incubationat 75° C. before binding RNA in the iron oxide system.

A volume of 600 uL plasma from BD PPT™ tubes was added to tubescontaining 40-45 mg iron oxide, and then 220 uL of 30 mM KPO₄ was addedto each of the tubes. ProK (6 units) was also added to each tube, andthe ProK was activated by incubating the tubes for 20 minutes either ina 55° C. water bath or in a 65° C. water bath. The tubes weretransferred to a 75° C. water bath and incubated for 20 minutes. Thesamples were cooled at room temperature for 5 minutes. Next, 180 uL of 6M glycine/HCl was added to each tube, and the samples were pipettemixed. Next, 10 uL yeast carrier RNA (10 ug/mL) was added to each tube,followed by 6 uL HIV stock RNA (10⁷ copies/ml) and pipette mixed. Theunbound samples were removed from the tubes by locking iron oxideparticles to the side of tube and aspirating the solution to waste. Theparticles were washed three times with 1000 uL wash solution (86 mMglycine/HCl) by pipette mixing, locking iron oxide particles to the sideof each tube, and aspirating unbound solution. The samples were theneluted by adding 400 uL elution buffer (90 mM Bicine, 50 mM KOH and 20mM KPO₄) to each tube and conducting 12 cycles of aspiration mixing,followed by 20 minutes of magnetic mixing at 60° C., followed by asecond round of 12-cycle aspiration mixing. Yeast carrier RNA (10 ug)was added to each tube. The target was added to pre-assay spiked controlsamples. The samples were removed to new tubes as described in Example 1and assayed by an HIV RT-SDA assay. The results are as follows:

Specimen Buffer Plasma Buffer Plasma Pro K   6 U   6 U   6 U   6 U Pro KActivation 55° C. 55° C. 65° C. 65° C. Pro K Incubation 75° C. 75° C.75° C. 75° C. Signal 49816 6182 28052 70729 52798 31904 74120 7394256186 53210 76484 58029 55034 57928 67257 51201 Mean Signal 53459 3730661478 63475 % CV 5 63 37 17

The pretreating of plasma with ProK at elevated temperatures (55° C. to65° C. activation temperature followed by 75° C. incubation temperature)demonstrates extraction efficiency as indicated by positive assaysignals.

Example 10 RNA Extraction from Whole Blood

This experiment was conducted to extract RNA from whole blood andexamine the use of additional wash steps to decrease carryover ofinhibitory components into the elution step.

Whole blood samples (500 uL) were each pretreated with 20 units of ProKand 300 uL of formamide for 20 minutes at 65° C. and 10 minutes at 85°C. One set of samples was pretreated with a 6-cycle wash for two washesand another set with a 9-cycle wash for three washes. The samples wereeluted with 85 mM KOH/75 mM Bicine, neutralized with 400 mM Bicine, anddetected with in the HIV gag gene RT-SDA amplification system. Theresults are as follows:

Wash 2X WASH and 6 CYCLES 3X WASH and 9 CYCLES COPIES/ML Signal MeanSignal Signal Mean Signal 0 64 241 0 35 151 0 27 474 0 119 61 57 2315,000 6798 1151 5,000 22268 2433 5,000 2831 4031 5,000 42 7985 45 191525,000 11154 30543 25,000 7470 53000 25,000 23408 42699 25,000 2011215536 39496 41435 50,000 9982 35527 50,000 47798 81777 50,000 1586227265 50,000 7165 20202 53345 49479

The results demonstrate that the additional wash improves assay signalindicating reduced carryover of inhibitory substances with increasedwashing.

While the invention has been described with some specificity,modifications apparent to those with ordinary skill in the art may bemade without departing from the scope of the invention. Various featuresof the invention are set forth in the following claims.

1. A method of treating a biological sample for extraction of nucleicacid therefrom comprising mixing the sample with at least one proteindenaturant and heating the mixture stepwise.
 2. The method of claim 1wherein the stepwise heating is in a temperature range of about 55° C.to about 85° C.
 3. A method of treating a biological sample forextraction of nucleic acid therefrom comprising treating the sample withat least one protein denaturant to form a reaction mixture and dilutingthe reaction mixture with a diluent.
 4. The method of claim 3 whereinthe diluent is selected from the group consisting of water, aqueousbuffer solutions and aprotic solvents.
 5. The method of claim 3 whereinthe method is carried out at a temperature at or above about 4° C. 6.The method of claim 5 wherein the method is carried out in a temperaturerange of about 25° C. to about 95° C.
 7. A reaction mixture for treatinga biological sample for extraction of nucleic acid therefrom, whereinsaid reaction mixture comprises at least one protein denaturant and atleast one aprotic solvent.
 8. The reaction mixture of claim 7 whereinthe protein denaturant is selected from the group consisting ofproteolytic enzymes, detergents, surfactants, solvents, amides, reducingagents, bases, protein denaturing salts and combinations thereof.
 9. Thereaction mixture of claim 8 wherein the protein denaturant is aproteolytic enzyme selected from the group consisting of proteinase K,pronase, pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase.10. The reaction mixture of claim 9 wherein the proteolytic enzyme isproteinase K.
 11. The reaction mixture of claim 8 wherein the proteindenaturant is a detergent selected from the group consisting of sodiumdodecyl sulfate, lithium dodecyl sulfate, polyethylene glycol sorbitanmonolaurate, polyethylene glycol sorbitan monooleate, NP-40, dodecyltrimethyl ammonium bromide, cetyl trimethyl ammonium bromide,3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, andpolyethylene glycol tert-octylphenyl ether.
 12. The reaction mixture ofclaim 8 wherein the protein denaturant is a surfactant.
 13. The reactionmixture of claim 8 wherein the protein denaturant is a solvent selectedfrom the group consisting of phenol, chloroform and isoamylalcohol. 14.The reaction mixture of claim 8 wherein the protein denaturant is anamide selected from the group consisting of N-ethylacetamide,N-butylacetamide and N,N-dimethylacetamide.
 15. The reaction mixture ofclaim 8 wherein the protein denaturant is a reducing agent selected fromthe group consisting of glutathione, β-mercatoethanol anddithiothreitol.
 16. The reaction mixture of claim 8 wherein the proteindenaturant is a base selected from the group consisting of KOH, NaOH,NH₄OH and Ca(OH)₂.
 17. The reaction mixture of claim 8 wherein theprotein denaturant is a protein denaturing salt selected from the groupconsisting of NaCl, KCl, LiCl, NH₄Cl, (NH₄)₂SO₄ and perchlorate salt.18. The reaction mixture of claim 7 wherein the aprotic solvent isselected from the group consisting of formamide, dimethylformamide,dimethyl sulfoxide, dimethylacetamide, acetronitrile, benzene, toluene,acetone, cyclohexane, n-heptane, sulfur dioxide andhexamethylphosphoramide.
 19. The reaction mixture of claim 18 whereinthe aprotic solvent is formamide.
 20. The reaction mixture of claim 7wherein the treatment is carried out at a temperature at or above about4° C.
 21. The reaction mixture of claim 20 wherein the treatment iscarried out in a temperature range of about 25° C. to about 95° C. 22.The reaction mixture of claim 20 wherein the treatment is carried out ina temperature range of about 70° C. to about 85° C.
 23. The reactionmixture of claim 7 wherein the treatment is carried out by stepwiseheating in a temperature range of about 55° C. to about 85° C.
 24. Thereaction mixture of claim 9 wherein the concentration of proteinase K isabout 1 to about 100 units per milliliter of biological sample.
 25. Thereaction mixture of claim 20 wherein the concentration of formamide isabout 10% to about 80% by volume.
 26. The reaction mixture of claim 7further comprising a solid support.
 27. The reaction mixture of claim 26wherein the solid support is selected from the group consisting of ironoxide, silica-coated particles, silica-coated membranes, glass fibermats, glass membranes, glasses, zeolites and ceramics.
 28. A nucleicacid extracted from a biological sample, wherein said nucleic acid isextracted by treating the sample with at least one protein denaturantand at least one aprotic solvent.
 29. The nucleic acid of claim 28wherein the protein denaturant is selected from the group consisting ofproteolytic enzymes, detergents, surfactants, solvents, amides, reducingagents, bases, protein denaturing salts and combinations thereof. 30.The nucleic acid of claim 29 wherein the protein denaturant is aproteolytic enzyme selected from the group consisting of proteinase K,pronase, pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase.31. The nucleic acid of claim 30 wherein the proteolytic enzyme isproteinase K.
 32. The nucleic acid of claim 29 wherein the aproticsolvent is selected from the group consisting of formamide,dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetronitrile,benzene, toluene, acetone, cyclohexane and n-heptane, sulfur dioxide andhexamethylphosphoramide.
 33. The nucleic acid of claim 32 wherein theaprotic solvent is formamide.
 34. The nucleic acid of claim 28 whereinthe nucleic acid is RNA.